Regulation of gene transcription in the ear is critical for normal development and audition. Eya4 is a member of the Eyes absent (eya) family of transcriptional co-activators that play critical roles in regulating gene expression in the ear. Mutations in the transcriptional co-activator EYA4 are responsible for autosomal dominant sensorineural hearing loss at the DFNA10 locus. Mice carrying mutant Eya4 alleles provide a model of human hearing loss. We have produced Eya4-deficient (Eya4+/- Eya4-/-) mice and Eya4-specific antibodies, reagents that will be used to define Eya4-target genes regulated during development and in post- natal life. Eya4-null mice (on the CBA background) are viable. Further, Eya4-/-mice, unlike Eya4+/- littermates and wildtype control mice have no measurable distortion product otoacoustic emission (DPOAE) response at 3 weeks post birth. Eya4+/- mice have significant deficiencies in the ability to detect low frequency sound by 22 weeks post birth;ongoing studies will determine if further deterioration of hearing occurs with age. The mechanisms responsible for hearing deficits in Eya4-/- mice appear to be complex. Eya4-/- ears have significant abnormalities of the stria vascularis and hair cells by 1-month post birth. In addition, Eya4-/- mice uniformly develop middle ear effusions and purulent otitis media by 6 weeks of age;neither occurs in wildtype or Eya4+/- mice. Because Eya4-/- middle ear effusions are sterile in mice treated with antibiotic from birth, we hypothesize that Eya4 plays a role in middle ear function. We propose here to define the role of Eya4 in inner and middle ears by studying Eya4-/- and Eya4+/- ears. Because Eya4 is a transcriptional co-activator, we hypothesize that Eya4 deficiency is mediated by altered transcription in Eya4-/- and Eya4+/- mice. We further hypothesize that by defining alterations in RNA levels in Eya4 deficient ears we will eventually identify the targets of Eya4 regulation and we will also define key components of molecular pathways required for normal inner and middle ear function. We have recently developed a novel method, termed PMAGE (polony multiple analysis of gene expression), for assessing RNA profiles that is 100X more sensitive than current methods. Application of this methodology to the study of wildtype and Eya4-deficient ears will provide new insights into transcriptional networks in the ear in general and, more specifically, into the role of Eya4. 4 specific aims are proposed: D1. Define anatomy and hearing in Eya4+/- and Eya4-/- ears. D2. Profile RNA expression in selected ear tissues of Eya4+/- and Eya4-/- mice using PMAGE. D3. Characterize Eya4-regulated genes and assess their role in ear development and hearing. D4. Define the molecules that mediate Eya4-/- induced otitis media. These studies should provide fundamental new information into the molecular basis for normal audition as well as hearing loss. In addition, these investigations may enlighten how gene-environment interactions increase otitis media susceptibility, a common contributor to human hearing loss.